The present invention concerns methods of transferring primordial germ cells to birds for the production of gametes therein. Such methods are useful in the conservation of endangered avian species, in reducing the time required to produce spermatozoa from slowly maturing species such as turkeys, decreasing the costs of maintaining breeder flocks, and altering the sex ratio of offspring flocks (e.g., to enhance the efficiency of production).
The ability to more easily produce gametes of particular avian species would be extremely useful to the avian veterinary and poultry production fields. For endangered species such as the whooping crane, it would be extremely useful to have a ready supply of male spermatozoa. For commercial birds such as turkeys, it would be desirable to more quickly and economically produce male spermatozoa. For meat-producing flocks, it is desirable to have ways to increase the ratio of male birds in the flock. Accordingly, there is a need for new ways to obtain avian spermatozoa.
Chimeras are composite organisms consisting of cells derived from more than one zygote. Experimental chimeras have been used to study cell to cell interaction and cell lineage analysis during development (A. McLaren, Mammalian Chimeras. Cambridge University Press, Cambridge (1976)). When chimeras are produced using material derived from very early embryos, organisms develop containing a full mixture of somatic tissues. If the starting material includes early germ cells or their precursors, the resulting individuals will produce gametes of both the donor and recipient genotypes. In addition, chimeras can be intraspecific, i.e. between two zygotes of the same species, or interspecific, i.e. between two different species.
Avian primordial germ cells (PGCs) like other vertebrate germ cells are extragonadal in origin and must undergo a complex journey to reach the gonad. The transfer of blastodermal cells and primordial germ cells has produced avian germline chimeras. Reynaud (J. Embryol. Exp. Morphol. 21:485-507 (1969)), a pioneer in the production of avian germline chimeras, reported the production of turkey-chicken germline chimeras by the intravascular transfer of dissociated turkey germinal crescent cells into previously sterilized chick embryos (accomplished by exposure of the recipient germinal crescent to ultra-violet light). PGCs obtained by mechanical dissociation of the endoderm of the germinal crescent (stage 5) were injected into the blood vessels of chicken embryos (3-5 days of incubation). Prior to injection the recipient embryos were sterilized at stage 8-10 (HandH) with ultraviolet light; however, the sterilization was not complete and caused problems with development and mortality. The turkey PGCs in the chick embryo were identified solely on the basis of their nucleo-cytoplasmic ratio. This method of identification was difficult and tenuous and could not be used for actively dividing turkey PGCs since the dividing germ cells gave an aberrant nucleo-cytoplasmic ratio.
In a succeeding study, the transferred PGCs were allowed to undergo maturation in the host gonads and apparently could give rise to gametes but they were not suitable for fertilization (Wilhelm, Roux""s Arch. Dev. Bio. 179:85-110 (1976)). The spermatozoa were incapable of fertilizing turkey eggs. They fertilized chick eggs but there was no normal development. Chicken spermatozoa were capable of activating the eggs obtained from female interspecific chimeras but they did not give rise to embryos. When the eggs were fertilized by turkey spermatozoa they developed into abnormal embryos that did not survive beyond stage 38 (HandH). Reynaud (J. Embryol. Exp. Morphol. 21:485-507 (1969)) used morphology as the only distinguishing characteristic in an attempt to identify turkey germ cells from chicken germ cells. Morphology alone is not sufficient for identifying chimeras and must be substantiated with other markers.
By reducing endogenous PGCs, the efficiency of generating germline chimeras, by repopulating the gonads with the desired donor PGCs, may be enhanced. A number of approaches to reduce PGCs have been utilized with varying degrees of success. Continuous exposure (20 days) to gamma irradiation (0.3-3.4 R/hr, 60Co) resulted in the complete destruction of oocytes at a dosage level of 3.4 and 1.8 R/hr (Mraz and Woody, Radiation Research 54:63-68 (1973)). However, hatchability was reduced at levels of 0.9 R/hr or higher. The application of continuous low-level gamma irradiation to reduce endogenous PGC is limited due to the relatively small numbers of eggs that can be exposed at any one time and the long period of exposure required.
Short-term exposure to a gamma source has also been attempted (Carsience et al., Development 117:669-75 (1993); Thoraval et al., Poultry Sci. 73:1897-1905 (1994); Maeda et al., Poultry Science 77:905-07 (1998)). In these studies, unincubated eggs were exposed to 500-700 rads just prior to the injection of stage X blastodermal or area pellucida cells. The incidence of germline chimerism following short-term gamma irradiation was highly variable. The basis for the inconsistent results were ascribed to xe2x80x9cdonor cells being injected into an inappropriate location . . . xe2x80x9d (Carsience et al., Development 117:669-75 (1993)).
Attempts to sterilize recipient embryos using ultraviolet light have been described (Reynaud, J. Embryol. Exp.Morphol. 21:485-507 (1969); Reynaud, J., Roux""s Archives of Developmental Biology 179:85-110 (1976); Aige-Gil and Simkiss (Brit. Poul. Sci. 32:427-438 (1991)). Aige-Gil and Simkiss concluded xe2x80x9cit is not possible to irradiate the germinal crescent, particularly at stage 4 of incubation, without inducing major abnormalitiesxe2x80x9d. The level of sterility appeared to be positively correlated with developmental abnormalities, thus limiting the practical use of UV-light as a means to reduce endogenous PGC.
The compound busulfan (1,4-butanediol dimethane sulfonate, BU) has been used as a chemotherapeutic agent in the treatment of leukemia (Bhagwatwar et al., Cancer, Chemotherapy and Pharmacology 37:401-08 (1996)). In 1963, Hemsworth and Jackson demonstrated that the administration of BU in rats could markedly impair the development of PGCs (Hemsworth and Jackson, J. Reproduction and Development 6:229-33 (1963)). Injection of BU into the yolk sac of chick embryos resulted in multiple malformations (Swartz, Teratology 21:1-8 (1980)). Hallett and Wentworth (Poultry Science 70:1619-23 (1991)) also report significant declines in hatchability following injection of an albumen suspension of BU into quail eggs. In some BU treated quail, there appeared to be an absence of germ cells in the gonads, while other similarly treated birds appeared normal. The authors suggested that xe2x80x9cinconsistencies in the delivery of BU to the embryoxe2x80x9d might explain the observed variation. They concluded that discovering a non-toxic solvent system would be necessary to eliminate the inconsistent results associated with use of a suspension. Aige-Gil and Simkiss (Brit. Poul. Sci. 32:427-438 (1991)) used saline or sesame oil suspensions of BU, or solublized BU in dimethyl sulphoxide (DMSO) in chick embryos. Administration of DMSO alone produced embryonic mortality, developmental delays, and malformations that exceeded those observed with saline. The teratogenic effects were greatly minimized when BU was suspended in sesame oil and injected into yolk. Injection of 100 xcexcg BU in sesame oil resulted in a sterility index of 95+%. In a subsequent experiment, Vick and co-workers (J. Reproduction and Fertility 98:637-41 (1993)) reported that the injection of 25, 50 and 250 xcexcg BU significantly reduced gonadal germ cells in chick embryos. They estimated that BU treatment increased the rate of germline chimerism 3.5-fold when compared to non-BU treated embryos. Bresler et al. (British Poultry Science 35:241-47 (1994)) demonstrated that treatment with BU and subsequent injection of PGCs could result in a significant repopulation of the gonad. Injection of 50 xcexcg BU, suspended in sesame oil reduced PGCs in the left and right gonad of 6 day-old chick embryos by 75 and 78%, respectively. Following the injection of a suspension of germinal crescent cells into BU-treated embryos, PGC numbers increased to 72 and 115% of controls for the left and right gonad, respectively.
The variability in delivery of BU to the gonad, and the resulting inconsistency in the effectiveness in reducing the number of PGCs, limits the usefulness of this technology.
Accordingly, there remains a need for new ways to accomplish the production and transfer of avian gametes.
A method for the production and collection of avian gametes comprises: reducing the number of primordial germs cells in a recipient avian subject in ovo; providing donor primordial germ cells from a donor avian subject; introducing the donor primordial germs cells into the recipient avian subject in ovo; incubating the recipient avian subject to hatch; and then raising the recipient avian subject to sexual maturity; wherein the recipient avian subject at sexual maturity produces gametes (e.g., sperm from male birds or ova from female birds) derived from the donor avian subject. In particular embodiments of the invention, the gametes are collected from the recipient avian subject. In other particular embodiments, the recipient avian subject is from a different species than the donor avian subject. For example, the donor avian species may be a whooping crane, and the recipient avian species may be a sand hill crane. In another example, the donor avian species may be a turkey, and the recipient avian species may be a chicken.
The production of turkey-chicken chimeras has wide applications. The transfer of male turkey PGCs is useful for turkey spermatogenesis in chicken gonads. This could accelerate spermatogenesis because the time required for production of sperm in chickens is 18 weeks as compared to 30 to 32 weeks in turkeys. The ability to culture PGCs and make germline chimeras could reduce the number of superior turkey sires currently needed to produce offspring. The ability to produce turkey sperm from a smaller and cheaper bird might also benefit the poultry industry.
The experimental chimeras could also provide a model to study the interaction between germ cells and somatic cells of different genotypes whereby it becomes possible to inquire whether its neighboring cells impose any of the germ cell characteristics upon it. This technique could also be utilized to transfer PGCs from low fecundity strains to more prolific birds, and for preserving PGCs in case of unexpected death or disease or in case an avian species is endangered under natural mating conditions (A. Tajima et al., Theriogenology 40:509-519 (1993)).
This aspect of the invention may also be practiced to increase the proportion of Z or W gametes produced by an avian subject. Typically, the inventive methods will be employed to increase the production of Z gametes (i.e., by the transfer of male, ZZ, PGCs). In birds, unlike mammals, it is the male that is the homogametic sex (ZZ) and the female which is the heterogametic sex (Zw). Therefore, in birds, it is the female that determines the gender of the offspring since she produces ova which carry either the Z or w chromosome. Thus, as noted below, by transferring male primordial germ cells (ZZ genotype) to female embryonic hosts, the percentage of Z-bearing ova produced by that host is increased and the percentage of male offspring is increased. An increase in the percentage of male offspring from broiler flocks is economically desirable for the corresponding greater feed conversion ratio and more efficient meat production so obtained.
Accordingly, a second aspect of the present invention is a method of increasing the proportion of male birds in a plurality of bird eggs, comprising: reducing the number of primordial germ cells in a female bird in ovo; introducing male (ZZ) avian primordial germ cells into the female bird in ovo; incubating the female bird to hatch; raising the female bird to sexual maturity; and then breeding the bird to produce a plurality of fertile bird eggs (with the ratio of male to female bird eggs produced from the bird being greater than that obtained in the absence of administering the male primordial germ cells to the bird in ovo). Typically, the method further comprises the step of incubating the plurality of bird eggs to hatch (with the ratio of male to female birds produced from the plurality of eggs being greater than that produced in the absence of administering the male primordial germ cells to the female bird in ovo). The female bird may be of any suitable species, such as chicken or turkey, and the primordial germ cells being administered are preferably from the same species as the female bird to which they are administered.
The foregoing and other objects and aspects of the present invention are explained in detail in the specification set forth below.